The nuclear factor of activated T cells (NF-AT) transcription factor family plays a pivotal role in T lymphocyte activation. NF-AT proteins regulate the T lymphocyte activation-dependent expression of several important cytokine genes, including IL-2, IL-3, IL-4, IL-5 and GM-CSF, as well as the expression of other immunologically important molecules such as CD40L and Fas. In concert, these gene products play a crucial role in regulating and coordinating the immune response. Understanding the mechanisms involved in the regulation of NF-AT will therefore provide important insights into the molecular mechanisms underlying the co-ordinate regulation of T lymphocyte activation genes and the initiation and regulation of the T lymphocyte immune response. NF-AT proteins appear to be regulated primarily by their subcellular localization. In resting T lymphocytes, NF-AT is located in the cytoplasm; however following antigen-receptor stimulation and the activation of calcineurin, NF-AT translocates from the cytoplasm to the nucleus, whereupon it can bind DNA and activate transcription. In aim 1, NF-ATc binding proteins implicated in the regulation of NF-ATc activity will be isolated and characterized. In aim 2, functionally important phosphorylation sites in NF-ATc will be identified and the role of these sites in the regulation of NF-ATc activity will be examined. In aim 3, the kinases responsible for phosphorylating functionally important NF-ATc residues in vivo will be identified and characterized.